| Objective:Professor Yongxian Cheng,dean of the school of pharmacy at Shenzhen University Health Science Center,created the formula for Shenjiang Yin on his own.Shenjiang Yin has a positive effect on reducing fatigue,according to preliminary experimental studies,but the exact mechanism is still unknown.A state of diminished physical or mental capacity brought on by sleep deprivation,circadian rhythm disruption,increased workload,etc.,fatigue is a complex phenomenon.According to clinical studies,patients with fatigue exhibit an imbalance in oxidative stress and energy metabolism.Energy metabolism is transferred through AMPK,which controls both autophagy and energy metabolism.The process of eliminating toxic byproducts of fatigue is comparable to autophagy,the scavenger’s method of cell death.One of the main factors affecting the quality of modern life is fatigue.Patients with fatigue are becoming more and more prevalent as pressure in contemporary society increases.In order to provide an experimental foundation for TCM fatigue prevention and treatment,the effects of Shenjiang Yin on the energy metabolism pathway AMPK/PGC-1α and autophagy in fatigue model mice were examined in this study.Methods:The fatigue model was established for this study to observe and record body weight,food intake,swimming time until exhaustion,etc.Pathology examined the general state of the mouse skeletal muscle tissue.The pathological conditions and alterations of the mouse skeletal muscle tissue were examined using H&E staining.The morphological and structural changes of gastrocnemius muscle and mitochondria were observed by transmission electron microscopy.The changes of reactive oxygen species(ROS)were observed by fluorescence staining.Lactic acid,urea nitrogen,and glycogen concentrations in mouse serum were found.Oxidation-stress markers’ malondialdehyde(MDA),glutathione peroxidases(GSH-Px),and superoxide dismutase(SOD)contents.AMPK,PGC-1,and m TOR,Beclin-1,and P62,which are associated with autophagy,were shown to have relative m RNA expression levels by q-PCR.The protein expression levels of PGC-1,AMPK,P-AMPK,Beclin,m TOR and P62 were detected.Chloroquine and AMPK inhibitors were modified for immunosuppression,and attention was paid to the changed differential proteins of Shenjiang Yin following intervention.Our theory was confirmed by PCR and Western Blot.Similar to before,the fatigue mouse model was repeated.The distinction was that,when compared to the model group and the Shenjiangyin intervention group,chloroquine and AMPK inhibitor were given for immunosuppression in order to observe and detect changes in the mice’s levels of fatigue,energy metabolism,oxidative stress,and general health.HE staining,electron microscopy and other pathological sections of skeletal muscle were observed.Results:(1)Shenjiangyin group’s exhaustive swimming time was longer than that of the model group(P<0.05);the levels of lactic acid and urea nitrogen were lower,the levels of glycogen were higher,the levels of glutathione peroxidase and superoxide dismutase were higher,and the levels of malondialdehyde were lower.Skeletal muscle edema and congestion are decreased,along with the inflammatory cell infiltration and tissue structure of the muscle.The amount of inflammatory cell infiltration and edema was lessened by H&E staining.Shenjiangyin intervention groups inhibited mitochondrial oxidative stress and decreased ROS levels in skeletal muscle cells when compared to the model group.The m RNA contents of AMPK and PGC-1,Beclin m RNA content of autophagy-related indexes,and the contents of m TOR and P62 were statistically significantly different from the model group in the dose groups of Shen Jiang Yin(P<0.01).Increases in the protein expression levels of AMPK and PGC-1,Beclin,the autophagy related index,and the protein expression levels of m TOR and P62 in comparison to the model group.(2)Compared with model group,Beclin m RNA and protein contents of autophagy related indexes were increased in drug administration group,while the contents of m TOR and P62 m RNA and protein were decreased(P<0.05).The exhaustive swimming time of the autophagy inhibitor chloroquine group was shortened(P < 0.05).The contents of lactic acid and urea nitrogen were increased,the content of glycogen was decreased,the contents of glutathione peroxidase and superoxide dismutase were decreased,and the content of malondialdehyde was increased(P < 0.05).The tissue structure of skeletal muscle was seriously damaged and inflammatory cell infiltration increased.Edema and congestion of skeletal muscle are aggravated.H&E staining showed increased infiltration and edema of inflammatory cells.The chloroquine inhibitor group aggravated mitochondrial destruction and increased ROS levels in skeletal muscle cells.(3)Compared with model group,m RNA and protein contents of AMPK and PGC-1αin Shenjiang Yin groups were significantly increased;EST was shortened in AMPK inhibitor group;The contents of lactic acid and urea nitrogen increased,the glycogen content decreased,the contents of glutathione peroxidase and superoxide dismutase decreased,and the contents of malondialdehyde and ROS increased(P < 0.05);Skeletal muscle was severely damaged and inflammatory cell infiltration increased(P <0.05).Edema and congestion of skeletal muscle are aggravated.H&E staining showed increased infiltration and edema of inflammatory cells.AMPK inhibitor group aggravated mitochondrial destruction in skeletal muscle cells.(4)Compared with autophagy inhibitors,there were no significant changes in AMPK and PGC-1α in inhibitor plus Shenjiangyin group.Compared with AMPK inhibitor group,m RNA and protein contents of Beclin autophagy related gene in AMPK inhibitor plus Shen Jiang Yin group were increased,while m TOR,P62 and protein contents were decreased(P<0.05).Conclusion:(1)Shenjiang Decoction can prolong the swimming time of tired mice,improve the fatigue condition of mice,reduce the damage of muscle fibers and mitochondria,improve the antioxidant capacity,maintain the homeostasis of energy metabolism and relieve fatigue.(2)Shenjiang Yin can regulate autophagy and AMPK/PGC-1α pathway to relieve fatigue,and autophagy changes after inhibition of AMPK/PGC-1α pathway.Therefore,it is suggested that Shenjiang Yin can regulate autophagy to relieve fatigue by activating AMPK/PGC-1α pathway. |
